Justin Chosich

Measurement of estradiol, estrone, and testosterone in postmenopausal human serum by isotope dilution liquid chromatography tandem mass spectrometry without derivatization.

BACKGROUND A high-throughput, sensitive, specific, mass spectrometry-based method for quantitating estrone (E1), estradiol (E2), and testosterone (T) in postmenopausal human serum has been developed for clinical research. The method consumes 100μl human serum for each measurement (triplicates consume 300μl) and does not require derivatization. We adapted a commercially available 96-well plate for sample preparation, extraction, and introduction into the mass spectrometer on a single platform. METHODS Steroid extraction from serum samples and mass spectrometer operational parameters were optimized for analysis of estradiol and subsequently applied to other analytes. In addition to determining the limit of detection (LOD) and limit of quantitation (LOQ) from standard curves, a serum LOQ (sLOQ) was determined by addition of known steroid quantities to serum samples. Mass spectrometric method quantitative data were compared to results using a state-of-the-art ELISA (enzyme-linked immunosorbent assay) using stored serum samples from menopausal women. RESULTS The LOD, LOQ, sLOQ was (0.1pg, 0.3pg, 1pg/ml) for estrone, (0.3pg, 1pg, 3pg/ml) for estradiol, and (0.3pg, 1pg, 30pg/ml) for testosterone, respectively. Mass spectrometry accurately determined concentrations of E2 that could not be quantified by immunochemical methods. E1 concentrations measured by mass spectrometry were in all cases significantly lower than the ELISA measurements, suggesting immunoreactive contaminants in serum may interfere with ELISA. The testosterone measurements broadly agreed with each other in that both techniques could differentiate between low, medium and high serum levels. CONCLUSIONS We have developed and validated a scalable, sensitive assay for trace quantitation of E1, E2 and T in human serum samples in a single assay using sample preparation method and stable isotope dilution mass spectrometry.

Joint MiRNA/mRNA Expression Profiling Reveals Changes Consistent with Development of Dysfunctional Corpus Luteum after Weight Gain

Obese women exhibit decreased fertility, high miscarriage rates and dysfunctional corpus luteum (CL), but molecular mechanisms are poorly defined. We hypothesized that weight gain induces alterations in CL gene expression. RNA sequencing was used to identify changes in the CL transcriptome in the vervet monkey (Chlorocebus aethiops) during weight gain. 10 months of high-fat, high-fructose diet (HFHF) resulted in a 20% weight gain for HFHF animals vs. 2% for controls (p = 0.03) and a 66% increase in percent fat mass for HFHF group. Ovulation was confirmed at baseline and after intervention in all animals. CL were collected on luteal day 7–9 based on follicular phase estradiol peak. 432 mRNAs and 9 miRNAs were differentially expressed in response to HFHF diet. Specifically, miR-28, miR-26, and let-7b previously shown to inhibit sex steroid production in human granulosa cells, were up-regulated. Using integrated miRNA and gene expression analysis, we demonstrated changes in 52 coordinately regulated mRNA targets corresponding to opposite changes in miRNA. Specifically, 2 targets of miR-28 and 10 targets of miR-26 were down-regulated, including genes linked to follicular development, steroidogenesis, granulosa cell proliferation and survival. To the best of our knowledge, this is the first report of dietary-induced responses of the ovulating ovary to developing adiposity. The observed HFHF diet-induced changes were consistent with development of a dysfunctional CL and provide new mechanistic insights for decreased sex steroid production characteristic of obese women. MiRNAs may represent novel biomarkers of obesity-related subfertility and potential new avenues for therapeutic intervention.

Omega-3 Fatty Acid Supplementation Lowers Serum FSH in Normal Weight But Not Obese Women

CONTEXT Dietary omega-3 fatty acids delay ovarian aging and promote oocyte quality in mice. OBJECTIVE To test whether dietary supplementation with omega-3 polyunsaturated fatty acids (PUFA) modulates reproductive hormones in reproductive-age women. DESIGN Prospective interventional study. SETTING Academic center. PARTICIPANTS Fifteen obese and 12 normal-weight (NW) eumenorrheic women, ages 28-34 years. INTERVENTION Two frequent blood-sampling studies were performed before and after 1 month of omega-3 PUFA supplementation with 4 g of eicosapentaenoic acid and docosahexaenoic acid daily. MAIN OUTCOME MEASURES Serum LH and FSH (basal and after GnRH stimulation). RESULTS The ratio of omega-6 to omega-3 PUFA was significantly reduced in plasma and red blood cell components for both groups after treatment (both P < .01). Omega-3 PUFA supplementation resulted in reduction of FSH and FSH response to GnRH by 17% on average (P = .06 and P = .03, respectively) in NW but not obese women. Serum levels of IL-1β and TNF-α were reduced after omega-3 PUFA supplementation (-72% for IL-1β; -56% for TNF-α; both, P < .05) in obese but not in NW women. This reduction, however, was not associated with a hormonal change in obese women. CONCLUSIONS Dietary administration with omega-3 PUFA decreased serum FSH levels in NW but not in obese women with normal ovarian reserve. This effect is intriguing and is directionally consistent with murine data whereby higher dietary omega-3 PUFA extends reproductive lifespan. Our results imply that this nutritional intervention should be tested in women with diminished ovarian reserve in an attempt to delay ovarian aging.

Evidence of GnRH Antagonist Escape in Obese Women

CONTEXT Assisted reproductive technology (ART) cycle cancelation rates are increased among overweight and obese women; however, the reasons for this are not completely clear. Premature luteinization due to inadequate endogenous gonadotropin suppression is a possibility for this higher risk of cancellation. OBJECTIVE The objective of the study was to investigate the impact of female obesity on the pharmacokinetics of cetrorelix (GnRH antagonist). DESIGN This was an interventional study. SETTING The study was conducted at a university clinical and translational research center. PARTICIPANTS Regularly menstruating obese (n = 10) and normal-weight (n = 10) women participated in the study. INTERVENTIONS A frequent blood sampling study was performed after a GnRH antagonist was administered, followed by recombinant LH. MAIN OUTCOMES MEASURED Pharmacokinetics of cetrorelix in obese vs normal weight women were measured. RESULTS Five of the obese women (50%) and none of the normal-weight women had a rebound of LH (defined as >50% increase in LH level from nadir) over the 14-hour postdose observation period. The obese group had a significantly decreased distributional half-life of cetrorelix compared with the normal-weight group (8.1 ± 1.6 vs 12.7 ± 6.2 hours, P = .02). The obese group exhibited increased clearance of cetrorelix compared with the normal-weight group (25.8 ± 6.8 vs 20.1 ± 8.3 L/h, P = .058). CONCLUSIONS The altered pharmacokinetics of cetrorelix in obese women may lead to premature ovulation during ART, and this could be one of the mechanisms that results in increased cycle cancelation in this group of women. In accordance with the higher gonadotropin requirements for obese women undergoing ART, weight-based dosing of GnRH antagonists may be required.

Adiposity Alters Genes Important in Inflammation and Cell Cycle Division in Human Cumulus Granulosa Cell

Objective: To determine whether obesity alters genes important in cellular growth and inflammation in human cumulus granulosa cells (GCs). Methods: Eight reproductive-aged women who underwent controlled ovarian hyperstimulation followed by oocyte retrieval for in vitro fertilization were enrolled. Cumulus GC RNA was extracted and processed for microarray analysis on Affymetrix Human Genome U133 Plus 2.0 chips. Gene expression data were validated on GCs from additional biologically similar samples using quantitative real-time polymerase chain reaction (RT-PCR). Comparison in gene expression was made between women with body mass index (BMI) <25 kg/m2 (group 1; n = 4) and those with BMI ≥25 kg/m2 (group 2; n = 4). Results: Groups 1 and 2 had significantly different BMI (21.4 ± 1.4 vs 30.4 ± 2.7 kg/m2, respectively; P = .02) but did not differ in age (30.5 ± 1.7 vs 32.7 ± 0.3 years, respectively; P = .3). Comparative analysis of gene expression profiles by supervised clustering between group 1 versus group 2 resulted in the selection of 7 differentially expressed genes: fibroblast growth factor 12 (FGF-12), protein phosphatase 1-like (PPM1L), zinc finger protein multitype 2 (ZFPM2), forkhead box M1 (FOXM1), cell division cycle 20 (CDC20), interleukin 1 receptor-like 1 (IL1RL1), and growth arrest-specific protein 7 (GAS7). FOXM1, CDC20, and GAS7 were downregulated while FGF-12 and PPM1L were upregulated in group 2 when compared to group 1. Validation with RT-PCR confirmed the microarray data except for ZFPM2 and IL1RL. As BMI increased, expression of FOXM1 significantly decreased (r = −.60, P = .048). Conclusions: Adiposity is associated with changes in the expression of genes important in cellular growth, cell cycle progression, and inflammation. The upregulation of the metabolic regulator gene PPM1L suggests that adiposity induces an abnormal metabolic follicular environment, potentially altering folliculogenesis and oocyte quality.

Luteal Phase Dynamics of Follicle Stimulating and Luteinizing Hormones in Obese and Normal Weight Women

Female obesity is a state of relative hypogonadotrophic hypogonadism. The aim of this study is to examine gonadotrophin secretion and response to gonadotrophin‐releasing hormone (GnRH) in the luteal phase of the menstrual cycle and to investigate the pharmacodynamics and pharmacokinetics of endogenous and exogenous luteinizing hormone (LH) in obese women.

Estradiol Priming Improves Gonadotrope Sensitivity and Pro-Inflammatory Cytokines in Obese Women.

CONTEXT Obesity is associated with a pro-inflammatory state and relative hypogonadotropic hypogonadism. Estrogen (E2) is a potential link between these phenomena because it exhibits negative feedback on gonadotropin secretion and also inhibits production of pro-inflammatory cytokines. OBJECTIVE We sought to examine the effect of estrogen priming on the hypothalamic-pituitary-ovarian axis in obesity. DESIGN, SETTING, AND PARTICIPANTS This was an interventional study at an academic center of 11 obese and 10 normal-weight (NW) women. INTERVENTION A frequent blood-sampling study and one month of daily urinary collection were performed before and after administration of transdermal estradiol 0.1 mg/d for one entire menstrual cycle. MAIN OUTCOME MEASURES Serum LH and FSH before and after GnRH stimulation, and urinary estrogen and progesterone metabolites were measured. RESULTS E2 increased LH pulse amplitude and FSH response to GnRH (P = .048, and P < .03, respectively) in obese but not NW women. After E2 priming, ovulatory obese but not NW women had a 25% increase in luteal progesterone (P = .01). Obese women had significantly higher baseline IL-6, IL-10, TGF-β, and IL-12 compared with NW (all P < .05); these levels were reduced after E2 (-6% for IL-1β, -21% for IL-8, -5% for TGF-β, -5% for IL-12; all P < .05) in obese but not in NW women. CONCLUSIONS E2 priming seems to improve hypothalamic-pituitary-ovarian axis function and systemic inflammation in ovulatory, obese women. Reducing chronic inflammation at the pituitary level may decrease the burden of obesity on fertility.

Use of a levonorgestrel-containing intrauterine system with supplemental estrogen improves symptoms in perimenopausal women: a pilot study.

Objective:The aim of this study was to compare perimenopausal symptomatology using a levonorgestrel-containing intrauterine system (LNG-IUS) + low-dose transdermal estradiol (TDE) with LNG-IUS alone. Methods:The trial was a double-blind, randomized, controlled pilot trial. Regularly cycling women aged 38 to 52 years, with at least one self-reported symptom (hot flashes, bloating, headache, adverse mood, or poor sleep), were randomized to either LNG-IUS + low-dose TDE gel (intervention) or LNG-IUS alone (control). TDE was administered once daily as a 0.06% gel containing 0.75 mg of TDE for 50 days. LNG-IUS was placed at least 90 days before TDE or placebo gel treatment to assure stable circulating LNG. Participants completed the Center for Epidemiologic Studies Depression scale (CESD), Hot Flash Related Daily Interference scale (HFRDIS), Pittsburgh Sleep Quality Index (PSQI), and Fatigue Severity Scale (FSS) at the time of LNG-IUS placement, at 90 days (the time of randomization to TDE/placebo), and 140 days (end of study). TDE and placebo groups were compared using repeated-measures analysis of variance. Results:Thirty-eight women aged 42.9 ± 2.7 years, with a mean BMI of 24.7 ± 3.3 kg/m2, were enrolled; 20 were randomized to TDE. Women receiving TDE had significantly improved FSS scores between days 90 and 140 (mean difference TDE: −0.8 ± 1.2 vs placebo: 0.1 ± 0.7; P = 0.026) and borderline significant improvement in HFRDIS scores (mean difference TDE: −5.5 ± 15.3 vs placebo: 4.2 ± 13.1; P = 0.076). Women who reported hot flashes at baseline and who received TDE had a significant decrease in HFRDIS scores between days 90 and 140 (n = 9, P = 0.035). CESD and PSQI scores were not associated with TDE use. Conclusions:A brief, low-dose estrogen intervention, combined with a LNG-IUS, led to significant improvement of some common perimenopausal symptoms. Such a “minimalist” approach to management of the perimenopause holds promise for reducing common, bothersome perimenopausal symptoms while maintaining effective contraception.

Acute recapitulation of the hyperinsulinemia and hyperlipidemia characteristic of metabolic syndrome suppresses gonadotropins

To determine the effect of lipid/heparin versus saline infusion, with or without concurrent euglycemic hyperinsulinemia, on serum follicle‐stimulating hormone (FSH) and luteinizing hormone (LH). Obesity is associated with hyperlipidemia, insulin resistance, and relative hypogonadotropic hypogonadism. It was hypothesized that acutely elevated fatty acids and insulin would impair gonadotropin secretion.

Corpus luteum as a novel target of weight changes that contribute to impaired female reproductive physiology and function

ABSTRACT Obesity and malnutrition are associated with decreased fecundity in women. Impaired reproductive capacity in obese women is often attributed to anovulation. However, obese women with ovulatory cycles also have reduced fertility, but the etiology of their impaired reproduction is only partially understood. Accumulating evidence suggests that obesity directly impairs oocyte and embryo quality as well as endometrial receptivity. In obese women, urinary progesterone metabolite excretion is decreased, but in excess of what can be explained by suppressed gonadotropin secretion, suggesting that apart from its central effect obesity may directly affect progesterone (P4) production. These observations have led to the novel hypothesis that obesity directly affects corpus luteum (CL) function. Similarly, we hypothesize that weight loss may contribute to luteal dysfunction. Here, we propose a non-human primate model, the vervet monkey, to examine the effect of weight gain and loss on menstrual cycle parameters and CL gene expression. In this model, weight gain and loss did not significantly alter menstrual cyclicity; however, both induced alterations in the CL transcriptome. In the weight gain monkey, we observed that impaired mid-luteal P4 secretion was associated with downregulation of steroidogenic pathways in CL. Collectively, these preliminary findings support our hypothesis that weight gain and loss may contribute to CL dysfunction. The vervet model described and preliminary observations provide a basis for a larger study to address this important question. Understanding the mechanisms by which weight gain and loss contribute to reproductive dysfunction can assist in the development of targeted treatments to enhance women’s reproductive capability when it is desired. Abbreviations: CL: corpus luteum; P4: progesterone; E2: estradiol; PDG: pregnanediol 3-glucoronide; LH: luteinizing hormone; FSH: follicle-stimulating hormone; GnRH: gonadotropin releasing hormone; BMI: body mass index; qrtPCR: quantitative real-time PCR; PGR: progesterone receptor; ART: assisted reproductive technology; IVF: in vitro fertilization; HPO: hypothalamic-pituitary-ovarian axis; MMPs: matrix metalloproteinases Gene symbols: LH receptor (LHGCR); cholesterol side-chain cleavage enzyme (CYP11A1); 3 beta-hydroxysteroid dehydrogenase type II (HSD3B2); steroidogenic acute regulatory protein (STAR); LDL receptor (LDLR); scavenger receptor B1 (SCARB1); ATP-binding cassette sub-family A member 1 (ABCA1); ATP-binding cassette sub-family G member 1 (ABCG1); apolipoprotein A (APOA1); 24 dehydrocholesterol reductase (DHCR24); 3-hydroxy-3-methylglytaryl-CoA reductase (HMGCR); vascular endothelial growth factor A (VEGFA); vascular endothelial growth factor C (VEGFC); vascular endothelial growth factor receptor 1 (VEGFR1); and TIMP metallopeptidase inhibitor 1 (TIMP1); amphiregulin (AREG); epiregulin (EREG); CCAAT/enhancer binding protein alpha (CEBPBA); cAMP responsive element binding protein 3-like 1 (CREB3L1); ADAM metallopeptidase with thrombospodin type 1 motif 1 (ADAMTS1); matrix metallopeptidase 9 (MMP9); cytochrome b-245 beta polypeptide (CYBB or NOX2); NADH oxidase (NCF2 or NOXA2); Fc fragment of IgG receptor IIb (FCGR2B); Fc fragment of IgG receptor IIb (FCGR2C); ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1); RAB27A member RAS oncofamily (RAB27A); hydroxyprostaglandin dehydrogenase (HPGD); prostaglandin-endoperoxidase synthase 1 (PTGS1); integrin B2 (ITGB2); leukotriene A4 hydrolase (LTA4H); radixin (RDX); ezrin (EZR); nuclear receptor subfamily 5 group A member 2 (NR5A2)